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Hepatitis C and B viruses in hepatitis B Surface antigen-negative hepatocellular carcinoma
GASTROENTEROLOGY
1992;103:1322-1327
Hepatitis C and B Viruses in Hepatitis B Surface Antigen-Negative Hepaiocellular Carcinoma JIN-CHUAN WEN-CHEN MING-YANG HSUAN-SHU
SHEU, GUAN-TARN HUANG, LING-NA SHIH, LEE, HUEY-CHI CHOU, JIN-TOWN WANG, PO-HUANG LAI, CHANG-YI WANG, PEI-MING YANG, LEE, and DING-SHINN
CHEN
Department of Internal Medicine, College of Medicine, National Taiwan University Republic of China; and United Biomedical Inc., Lake Success, New York
The relative role of hepatitis C virus and hepatitis B virus in hepatitis B surface antigen-negative hepatocellular carcinoma was evaluated by polymerase chain reaction in 31 patients from Taiwan. Twenty-one were positive for antibody to hepatitis C virus (group 1)and 10 were negative (group 2). Of the group 1 patients, hepatitis C viral RNA was detected in the serum by polymerase chain reaction in 16 and in the liver tissue in 17, whereas hepatitis B viral DNA was found in the liver tissue in only 4, and none were found in the serum. In group 2 patients, hepatitis C viral RNA was detected in the serum of 1 and in the liver tissue of another. In contrast, hepatitis B viral DNA was found in the serum of 4 patients and in the liver tissues of 5. It was concluded that hepatitis C virus plays an important role in hepatocarcinogenesis in hepatitis B surface antigen-negative patients in Taiwan, especially in those who had antibody to hepatitis C virus; in those without antibody to hepatitis C virus, hepatitis B virus might still be associated with the development of hepatocellular carcinoma in a significant proportion of such patients. epatocellular carcinoma (HCC) is one of the most common malignancies in the world, especially in sub-Saharan Africa and Southeast Asia.ln’ Chronic hepatitis B virus (HBV) infection has been well documented to have a major role in the development of this cancer.‘s3 In patients negative for hepatitis B surface antigen (HBsAg), integration of HBV DNA sequences in the HCC tissue has been found.4-7 However, the positive rate reported has been inconsistent.4-8 Recently, the hepatitis C virus (HCV) has been implicated as another major causative agent of HCC in areas where HBV infection is low in prevalence.g-ll In HBV hyperendemic areas such as Taiwan, the prevalence of antibody to HCV (anti-HCV)
H
LEE,
Hospital, Taipei, Taiwan,
has been found to be much higher amongHBsAg-negative HCC patients than among HBsAg-positive HCC patients.” However, the significance of the association of HCV with HCC is difficult to interpret from serologic markers alone because although they are not antigenemic for HBsAg, nearly all of the HBsAg negative HCC patients in Taiwan have been infected by HBV as shown by the presence of antibodies to surface (anti-HBs) or core (anti-HBc) antigen.3 The polymerase chain reaction (PCR), which can detect a single copy of gene, provides a sensitive method to detect the HCV and HBV genome.13-l* To clarify the relative role of HCV and HBV in HBsAg-negative HCCs, we used PCR to detect the HCV RNA and HBV DNA sequences in the serum and liver tissues from 31 HBsAg-negative HCC patients in Taiwan. Patients and Methods Patients The 31 subjects selected for this study were taken from a total of 357 HCC patients seen consecutively in a period of one year and 6 months (July 1988-1990)in our clinic. Of them, 258 were positive for HBsAg and 99 were negative. Among the 99 HBsAg-negative patients, 31 who had frozen liver tissues and serum samples available for analysis were included in this study. The 31 patients ranged from 27 to 78 years old (mean 60), with 19 males and 12 females. Antibodies to HCV were identified in 21 of these 31 cases (67.7%; Table l),15 by standard assays for anti-C160 anti-HCV enzyme-linked immunosorbent assay (Ortho Diagnostic Systems, Raritan, NJ). An additional 6 anti-HCV positive cases were identified by a more sensitive second-generation anti-HCV immunoassay, (UBI HCV EIA; United 0
1992 by the American Gastroenterological 0016-5065/92/$3.00
Association
HCV AND HBV IN HBsAc-NEGATIVE HEPATOMA
October 1992
Table 1. Clinical Data and Results of HCV RNA and HBV DNA in HBsAg-Negative
HCC Patients
1323
Who Were Positive for
Anti-HCV (Group 1) HBV DNA by PCR
HCV RNA by PCR
HBV DNA by Tumor
Se1lllIl
Nontumor
Tllm0r
Nontumor
Southern blot
Tumor Case
Liver
Anti-
Anti-
Size
Primer”
state
HBs
HBc
km)
I
Primer”
I
II
Negative
II
1
CIR
P
N
6.2
P
P
N
N
2
CIR
P
P
2.4
P
P
P
P
Primer”
strandb
I
strandb
P
P
P
P
N
N
Pre-
PE-
Negative
II
Serum
S
S
Core
x
s
s
NonCore
X
Tumor
tumor
N
N
N
N
NN
N
N
N
N
N
N
N
N
N
NN
N
N
N
N
N
3
CIR
P
P
2.0
P
P
P
P
P
P
P
P
N
N
N
N
NN
N
N
N
N
N
4
CIR
P
P
2.9
P
P
P
P
N
P
P
N
N
N
N
N
NN
N
N
N
N
N
P
P
P
N
N
N
N
NN
N
N
N
N
N
P
P
P
P
N
N
N
N
NN
N
P
N
N
N N
5
CIR
P
P
2.4
P
P
N
N
6
CIR
P
P
2.6
P
P
P
P
7
CIR
P
P
2.4
P
P
P
P
P
P
P
P
N
N
N
N
NN
N
N
N
N
8
CIR
N
P
5.5
P
P
P
P
P
P
P
P
N
N
N
N
NN
N
N
N
N
N
9
CIR
P
P
5.4
P
P
P
P
P
P
P
P
N
P
P
N
NN
P
N
N
P
N
10
CIR
P
P
2.7
P
P
P
P
N
P
P
P
N
N
N
P
NN
N
N
N
N
N
11
CIR
P
P
3.6
P
P
P
N
P
P
N
P
N
N
N
N
NN
N
N
N
N
N
12
CIR
N
N
9.1
P
P
N
N
P
P
P
N
N
N
N
NN
N
N
N
N
N
13
CIR
P
P
3.0
P
P
P
P
P
P
P
N
N
N
N
NN
N
N
N
N
N
14
CIR
N
P
2.3
N
N
N
N
N
N
N
N
N
N
NN
N
N
N
N
N
P
N
N
N
N
NP
N
P
N
N
N
N
N
N
N
N
NN
N
N
N
N
N
N
N
N
N
NN
N
N
N
N
N
N
N
N
N
N
N
NN
N
N
N
N
N
P
P
P
N
N
N
N
NN
N
N
N
N
N
N
N
N
N
NN
N
N
N
N
N
N
N
N
N
NN
N
N
N
N
N
15
CIR
N
P
1.5
N
N
N
N
N
N
16
CIR
P
P
2.2
N
N
N
N
P
N
17
CIR
N
N
N
N
N
N
N
N
18
CIR
P
P
4.1
N
N
P
N
P
P
19
CIR
N
P
3.3
P
P
P
P
P
>lO
20
CPH
P
P
3.4
P
P
N
N
N
N
21
CIR
N
P
1.4
P
P
N
N
P
N
P
CIR, cirrhosis; CPH. chronic persistent hepatitis; N, negative: P, positive. “Result of HCV positive strand RNA. Primer 1: primer derived
from 5’noncoding
region. Primer II: primer from nonstructure
region
bResult of negative strand HCV RNA. Only cases with positive strand HCV RNA were tested.
Biomedical, Inc., Lake Success, NY), which incorporated both nonstructural and capsid antigen for antibody capture.lg The 21 HCC patients with seroreactivity to HCV were designated group 1 (Table 1). The remaining 10 HCC patients were negative for antiHCV and were placed into group 2 (Table 2). Sera of the 31 HBsAg-negative HCC subjects were also tested for the presence of anti-HBs and anti-HBc (Ausria-II, Abbott Laboratories, North Chicago, IL). All but 2 patients were positive for either HBV marker, most of them being positive for both (Tables 1 and 2). The 2 patients negative for both antibody
markers of HBV infection were each positive for anti-HCV. Of the 21 anti-HCV positive patients, 20 had liver cirrhosis, whereas only 3 of the 10 negative HCC subjects were cirrhotic (Tables 1 and 2). HCV RNA Serum sample from each patient as well as liver tissues from both tumor and nontumor portion were evaluated by PCR to detect the HCV RNA and HBV DNA sequences. RNA for HCV PCR analysis was extracted from serum as described previously.‘7~‘8 RNA of tissue was isolated from minced
Table 2. Clinical Data and Results of HCV RNA and HBV DNA in HBsAg-Negative Anti-HCV (Group 2)
HCC Patients
HCV RNA by PCR
Who Were Negative for
HBV DNA by PCR HBV DNA by
Serum
TUIIIOI
Nantumor
Tllm0r
Nontumor
Southern blot
Tumor Case
Liver
Anti-
Anti-
Size
state
HBs
HBc
km1
Primer’ I
PrimeP
II
I
Negative II
Primer”
strandb
I
II
Negative strandb
PESerum
s
s
22
CIR
P
P
2.4
N
N
N
N
N
N
NNNNNNNNNN
23
CPH
P
P
11.5
N
N
N
N
N
N
PPP
24
NORM
P
P
>lO
N
N
N
N
N
N
PPP
25
CIR
P
N
>lO
N
N
N
N
N
N
N
26
CPH
P
P
7.0
N
N
N
N
27
NORM
P
P
19.0
N
N
P
N
26
FM
P
P
N
9.4
N
N
P
N
N
P
N
P
P
X
Tumor
tumor
P
N
N
N
NNN
PP
P
N
NN
N
N
NNNN
NN
N
N
NNNN
NN
N
N
NNNN
NN
N
N
N
N
NNN
P
P
6.3
N
N
N
N
N
N
NPNPPPNPPP
P
P
4.5
P
P
N
N
N
N
PNNPNNNNNN
31
CPH
P
P
14.0
N
N
N
N
N
N
P
from 5’noncoding
NonCore
NNN
NORM
NN
region. Primer II: primer from nonstructure
N N N
NNNN
normal; N. negative; P. positive.
bResults of negative strand HCV RNA, only cares with positive strand HCV RNA were tested.
s
NNN
CIR
“Results of positive strand HCV RNA. Primer I: primer derived
s
PP
PPPP P
29
NORM,
x
PPPP
30
CIR. cirrhosis: CPH, chronic persistent hepatitis: FM, fatty metamorphosis;
PWCore
region.
NN
N
N
1324
SHEU ET AL.
liver by a single step extraction thiocyanate-phenol-chloroform.”
GASTROENTEROLOGY
with guanidinium
Positive Strand HCV RNA For the detection of positive strand HCV RNA, RNA from 20 p,L serum or 1.0 pg tissue was reverse transcribed to complementary DNA (cDNA) using 2 sets of antisense primer (primer HCJ296R: SCACTCGCAAGCACCCTATCAGGCAGTACCAJ and primer JK3966R: 5’ATCCCGCTGATGAAGTTCCACATGTGCTTC3’) at 37°C for 90 minutes. Two pairs of primers were used for HCV PCR analysis, one pair from the 5’noncoding regions (primer HCJ004:5’CACTCGCAAGCACCCTATCAGGCAGTACCA3’ and primer HCJ296R as above); the other set from a deduced nonstructural protein encoding region (primer JK3779:5’GAGTGCGCCTCACACCTTCCTTACATCGAAY and primer JK3966R as above). Detection of the HCV amplification products was done on Southern transfers, with 2 labeled 30 oligonucleotides specific for the 5’ noncoding and nonstructural protein sequences (probe HCJ131: YCTGCGGAACCGGTGAGTACACCGGAATTGCY and probe C3821P30:5’GCTCGCCGAGCAGTTCAAGCAGAAGGCCCT3’ respectively). Southern transfer, hybridization, and autoradiography were performed as described previously.17,‘8 Negative Strand HCV RNA To evaluate the status of HCV replication in the liver, liver samples that contained positive strand HCV RNA were further assayed for the presence of negative strand HCV RNA.Z’ RNA from liver tissue was first reverse transcribed with the sense primer derived from the 5’noncoding region (primer HCJ 004 as above). To completely inactivate the reverse transcriptase, the mixture was heated at 98°C for 2 hours. The PCR was then performed as above using the primer pairs derived from 5’noncoding region as above (HCJ 004 and HCJ 296R). HBV DNA PCR assay. For HBV DNA analysis, extraction of serum DNA was done,17,18 and the tissue DNA was extracted with classical method as described before.7 For serum HBV PCR, a pair of nucleotide specific for the core region was used as primer and a 30-mer 32P-labeled oligonucleotide as probe.‘7,‘8 For tissue DNA, 4 pairs of primers specific for core, pre-S, S, and X genes of HBV were used (Table 3). The PCR products were identified after Southern transfer with each of the 4 specific 30-mer oligonucleotides (Table 3). Southern blot analysis. To analyze the status of HBV DNA in the liver, all the tissue DNA samples
Table
3.
Vol. 103, No. 4
Primers and Probes User for Detection DNA by PCR in Tissues
Pre-S region primer 5’GGG TCA 5’ TTC CTG probe 5’GCA AAT S region primer 5’TCT TCT 5’ CTG AAC probe 5’CGG AAA Core region primer 5’ TTT GGT 5’AGA TTC
of HBV
CCA TAT TCT TGG GA 3’ ACT GGC GAT TGG T 3’ CCA GAT TGG GAC TTC AAT CCC AAC 3’
TGT TGG TTC TTC TGG ACT ATC AAG 3’ AAA TGG CAC TAG TAA ACT GAG CCA 3’ TTG CAC CTG TAT TCC CAT CCC ATC 3’
GTC TTT CGG AGT GTG GAT TCG CAC 3’ CCG AGA TTG AGA TCT TCT GCG ACG 3’
probe 5’GAC CAC CAA ATG CCC CTA TCC TAT CAA CAC 3’ X region primer 5’ATG GCT GCT AGG CTG TGC TGC CAA CTG GAT 3’ 5’ CAA GAG TCC TCT TAT GTA AGA CCT TGG GCA 3’ probe 5’ TGC ACT TCG CTT CAC CTC TGC ACG TCG CAT 3’
from the 31 patients were also subjected to Southern blot analysis. Ten micrograms of purified DNA was digested with Hind III, EcoRI, BamHI or StyI, and electrophoresed on 0.8% agarose gel. Southern transfer and hybridization using a 32P-labeled whole genomic HBV DNA as probe was performed as described previously.7 Results HCV RNA Positive stranded HCV RNA. Among the 21 anti-HCV positive group 1 patients, positive strand HCV RNA was detected in the serum in 16 (76.2%) by both sets of HCV primers. Positive stranded HCV sequences were also identified in the liver tissues of 17 (80.9%) of the group 1 patients, 12 in the HCC part and 16 in the nontumor part (Table 1,Figure 1).HCV RNA could be found in the serum, tumor, and nontumor liver tissues of 10 anti-HCV positive cases, in the serum and tumor alone of only 1 case, and in the sera and nontumor liver tissues alone of 3 patients, including those of case 12 who were negative for all HBV serological markers. There were 2 anti-HCV positive patients with no HCV sequences detectable by PCR. HCV RNA was found in only 1 of the 10 anti-HCV negative serum samples of group II (Table 2, case 30), and none could be found in the tissue samples of that individual. Only 1 group 2 patient was positive for HCV RNA in the liver tissues, with HCV sequences
October 1992
012345678
HCV AND HBV IN HBsAG-NEGATIVE
0:
HEPATOMA
1325
012345678
Figure 1. Detection of HCV RNA in the liver tissues by PCR using 5’noncoding region as primer. Arrow indicates 293 base pairs. Lane 0: &xDNA/HaeJII fragment; lanes Z-3: 5 X lo’, 5 X 10,and 5 copies of cloned HCV cDNA fragment; lane4: negative control; lanes 5 and 6: patient 8 in group 1,nontumor and tumor part; lanes7 and 8:patient 14 in group 1,nontumor and tumor part. (A)Gel electrophoresis after PCR amplification showing positive bands in lanes 1, 2, 5, and 6. Lane 6 is faintly visible. (B)Autoradiography showing positive bands in lanes 1, 2, 3, 5, and 6.
identified in the tumor and nontumor liver tissue (case 27). Altogether, among the 31 HBsAg-negative HCC patients of group 1 and 2, HCV positive strand RNA was detected in either the serum or liver tissue in 20 (64.5%) and in the tumor and/or nontumor tissues of 18 (58.1%). The primer sets in PCR for detection of HCV RNA displayed a slight difference in sensitivities. Both sets of the primers amplified HCV sequences in all 17 of the PCR positive serum samples; while the 5’noncoding region primers detected HCV RNA in all 17 of the PCR-positive nontumor tissue samples, the nonstructural protein region primers distinguished 12 of the 17; and the 5’noncoding region primers identified 13 tumor samples as PCR-positive, whereas the nonstructural protein set amplified only 10 of these tumor samples (Tables 1 and 2). Negative strand HCV RNA. In group 1 patients who had positive strand HCV RNA in the liver tissue, the negative strand RNA was detected in 10 of the 12 tumor specimens (83.3%) and in 13 of the 16 nontumor counterparts (81.3%) (Table 1). In group 2 patients, the negative strand HCV RNA was also found in the liver tissues (both the tumor and nontumor part) of the only patient whose tissue had positive strand HCV RNA (case 27, Table 2). HBV DNA PCR assay. By the HBV PCR assay, none of the 21 anti-HCV positive group 1 patients had HBV DNA in the serum (Table 1). However, 4 (19.0%)
were found to have HBV DNA sequences in the liver tissue, 3 in the nontumor portion and 2 in the tumor portion with one (case 9) in both HCC and nontumor part. Of the 4 patients positive for HBV DNA in the tissue, 3 also had HCV RNA in the serum and in the liver tissues, whereas the remaining one had no HCV sequence in either the serum or liver tissue. Of the group 2 anti-HCV negative patients, 4 (40%) were found to have HBV DNA in the serum. HBV DNA sequences were also identified in the liver tissues in 5 cases (50%) with 3 in both tumor and nontumor part and 2 in the tumor part alone (Figure 2). Among the 4 patients who had HBV DNA in the serum, 2 (cases 23 and 24) had HBV DNA in both the tumor and nontumor part detected by all 4 HBV primer sets (Table 2); another one (case 30) had HBV DNA in the tumor part detectable only by primers for HBV core sequences. The remaining one (case 31) had no HBV sequences in the liver tissue. On the other hand, 2 patients (cases 25 and 29) who had no HBV DNA in their serum had detectable HBV sequences in the liver tissues (1 in the tumor alone and the other in both the tumor and nontumor portions). Thus persistent HBV infection as evidenced by HBV DNA in either serum or liver tissue was present in 6 (60%) of these HBsAg-negative anti-HCV negative patients. Overall, among the 31 HBsAg-negative HCC patients of groups 1 and 2, HBV DNA sequences were detected in the liver tissue of 9 cases (29.0%) and in the serum and/or tissues of 10 (32.3%). Southern blot analysis. By Southern blot analysis, only one group 1 patient (case 9; Table 1) and 3
1326
SHEU ET AL.
0123456
GASTROENTEROLOGY
Vol. 103. No. 4
0123456
Figure 2. Detection of HBV DNA in the liver tissue by PCR using HBV core sequence as primer. Arrow indicates 190 base pairs. Lane 0: 4xDNA/HaeIII fragment; lanes 2 and 2: patient 23 in group 2, tumor and nontumor part; lanes 3 and 4: patient 30 in group 2, tumor and nontumor part; lanes 5 and 6: patient 31 in group 2, tumor and nontumor part. (A)Gel electrophoresis after PCR amplification showing positive bands in lanes 1 and 2. (B) Autoradiography showing positive bands in lanes 1-3.
group 2 patients (Cases 23,24,and 29; Table 2) had detectable HBV genome in the tumor part and no HBV DNA was found in the nontumor part. All the HBV DNA was present in integrated form. Three of the patients showed only one discrete band, whereas the remaining one (case 23;Table 2)showed multiple discrete bands. Discussion We found that HCV RNA was detected in either the serum, tumor and/or nontumor tissues of 20 of the 31 non-HBsAg antigenemic HCC patients, associating persistent HCV infection with 64.5% of those cases. The correlation of chronic HCV infection with HCC was more marked in the 21 HBsAg-negative anti-HCV positive patients. HCV RNA was found in either the serum, liver, or tumor samples of 18 of these 21 (85.7%). Although the PCR method could not differentiate between tissue derived and serum derived HCV, we found that the majority (>80%) of group 1 patients who had positive strand HCV RNA in the liver tissue also had negative strand HCV RNA in the liver tissue. Indeed the presence of the replicative form of HCV RNA proved that active HCV infection is present in these patients. Moreover, the low prevalence of HBV sequence in these 21 HCC patients is in favor of chronic HCV infection to be etiologically important for HCC in the HBsAg negative anti-HCV positive patients. On the other hand, 4 patients in this group also had HBV DNA sequences in the tissue; 3 of them also had concurrent HCV RNA in the tissue and serum. This finding indicates that simultaneous persistent infection of HCV and HBV
might contribute to the hepatocarcinogenesis in some of these HBsAg negative HCC patients. In the group 2 anti-HCV negative patients, HCV RNA was uncommonly found, being present in the serum of only one and in the liver of another. In contrast, HBV DNA was more frequently found in either the serum or tissue (60%).By Southern blot hybridization, 3 patients in this group had an integrated form of HBV sequences in the tumor part. This finding indicated that in these HBsAg negative antiHCV negative HCC patients, HBV might still play a more important role than HCV in the hepatocarcinogenesis. HBV DNA was not detectable in the patient (Case 27;Table 2) who had both positive and negative strands of HCV RNA in the liver tissue, suggesting HCV might still be associated with the development of HCC even in the absence of circulating anti-HCV antibody. By Southern blot hybridization, most investigators reported that only a minority of the HBsAg negative HCC had HBV DNA integration in the tumor cells, although a very high positive rate has been describede4s5 Consistent with our previous data,7 we again showed a rather low positive rate in this setting of HCC patients. Although the positive rate of HBV DNA detected by Southern blot analysis is much lower than those by PCR as shown in our study in the tumor tissue, the fact that they all appear in integrated form and not in the nontumor part suggests that the HBV is present in more copies in the tumor part and might well represent the clonal expansion of HBV-integrated hepatocytes in the cancer tissue. In both groups of HCC patients (Tables 1 and 2), the primer pair corresponding to the 5’ noncoding se-
October 1992
quence of the HCV genome was more sensitive than the nonstructural protein region primers for the detection of HCV RNA. This observation is in agreement with nucleic acid sequence data showing the 5’ noncoding region to be more conserved than the nonstructural region.” We conclude that HCV plays an important role in the hepatocarcinogenesis in HBsAg-negative subjects in Taiwan, especially in those positive for antiHCV; in the HBsAg negative anti-HCV negative HCC patients, chronic HBV infection might still be closely associated with the development of HCC. References 1 Linsell DA, Higginson J. The geographic
2.
3
4.
5.
6.
7.
8.
9.
10.
11.
pathology of liver cell cancer. In: Cameron HM, Linsell DA, Warwick GP, eds. Liver cell cancer. New York: Elsevier, 1976:1-16. Beasley RP. Hepatitis B virus as the etiologic agent in hepatocellular carcinoma-epidemiologic considerations. Hepatology 1982;2(Suppl):21-26. Chen DS. Hepatitis B virus infection, its sequelae, and prevention in Taiwan. In: Okuda K, Ishak KG, eds. Neoplasms of the liver. Tokyo: Springer-Verlag, 1987:71-80. Brechot C, Pourcel C, Hadchouel M, Dejean A, Louis A, Scotto J, Tiollais P. State of hepatitis B virus DNA in liver diseases. Hepatology 1982;2(Suppl):27-34. Brechot C, Nalpas B, Courouce AM, Duhamel G, Callard P, Carnot F, Tiollais P, Berthelot P. Evidence that hepatitis B virus has a role in liver-cell carcinoma in alcoholic liver disease. N Engl J Med 1982;306:1384-1387. Imazeki F, Omata M, Yokosuka 0, Okuda K. Integration of hepatitis B virus DNA in hepatocellular carcinoma: reappraisal after exclusion of possible bacterial contamination. Cancer 1986;58:1055-1060. Lai MY, Chen PJ, YangPM, Sheu JC, Sung JL, Chen DS. Identification and characterization of intrahepatic hepatitis B virus DNA in HBsAg-seronegative patients with chronic liver disease and hepatocellular carcinoma in Taiwan. Hepatology 1990;12:575-581. Paterlini P, Gerken G, Nakajima E, Terre S, D’Errico A, Grigioni W, Nalpas B, Franc0 D, Wands J, Kew M, Pisi E, Tiollais P, Brechot C. Polymerase chain reaction to detect hepatitis B virus DNA and RNA sequences in primary liver cancers from patients negative for hepatitis B surface antigen. N Engl J Med 1990;323:80-85. Ohkoshi S, Kojima H, Tawaraya H, Miyajima T, Kamimura T, Asakura H, Satoh A, Hirose S, Hijikata M, Kato N, Shimotohno K. Prevalence of antibody against non-A, non-B hepatitis virus in Japanese patients with hepatocellular carcinoma. Jpn J Cancer Res 1990;81:550-553. Hasan F, Jeffers LJ, De Medina M, Reddy KR, Parker T, Schiff ER, Houghton M, Choo QL, Kuo G. Hepatitis C-associated hepatocellular carcinoma. Hepatology 1990;12:589-591. Saito I. Miyamura T, Ohbayashi A, Harada H, Katayama T, Kikuchi S, Watanabe Y, Koi S, Onji M, Ohta Y, Choo QL, Houghton M, Kuo G. Hepatitis C virus infection is associated
HCV AND HBV IN HBsAG-NEGATIVE
HEPATOMA
1327
with the development of hepatocellular carcinoma. Proc Nat1 Acad Sci USA 1990;87:6547-6549. 12. Chen DS, Kuo GC, Sung JL, Lai MY, Sheu JC, Chen PJ, Yang PM, Hsu HM, Chang MH, Chen CJ, Hahn LC, Choo QL, Wang TH, Houghton M. Hepatitis C virus infection in an area hyperendemic for hepatitis B and chronic liver disease: the Taiwan experience. J Infect Dis 1990;162:817-822. 13. Wang JT, Wang TH, Lin JT, Sheu JC, Chen DS. Hepatitis C virus RNA in saliva of patients with post-transfusion hepatitis C infection (letter). Lancet 1991;337:48. 14. Garson JA, Tedder RS, Briggs M, Tuke P, Glazebrook JA, Trute A, Parker D, Barbara JA, Contreras M, Aloysius S. Detection of hepatitis C viral sequences in blood donations by “nested” polymerase chain reaction and prediction of infectivity. Lancet 1990;335:1419-1422. 15. Ohkoshi S, Kato N, Kinoshita T, Hijikata M, Ohtsuyama Y, Okazaki N. Ohkura H, Hirohashi S, Honma A, Ozaki T, Yoshikawa A, Kojima H, Asakura H, Shimotohno K. Detection of hepatitis C virus RNA in sera and liver tissues of non-A, nonB hepatitis patients using the polymerase chain reaction. Jpn J Cancer Res 1990;81:862-865. 16. Yoneyama T, Takeuchi K, Watanabe Y, Harada H, Ohbayashi A, Tanaka Y, Yuasa T, Saito I, Miyamura T. Detection of hepatitis C virus cDNA sequence by the polymerase chain reaction in hepatocellular carcinoma tissues. Jpn J Med Sci Biol 1990;43:89-94. 17. Shih LN, Sheu JC, Wang JT, Huang GT, Chen JS, Sung JL, Wang TH, Chen DS. Detection of hepatitis B viral DNA by polymerase chain reaction in patients with hepatitis B surface antigen. J Med Virol 1990;30:159-162. 18. Shih LN, Sheu JC, Wang JT, Huang GT, Lee HS, Sung JL, Wang TH, Chen DS. Serum HBV DNA in healthy HBsAg-negative Chinese adults evaluated by polymerase chain reaction. J Med Virol 1990;32:257-260. 19. Hosein B, Fang CT, Popovsky MA, Ye J, Zhang M, Wang CY. Improved serodiagnosis of hepatitis C virus infection with synthetic peptide antigen from capsid protein. Proc Nat1 Acad Sci USA 1991;88:3647-3651. 20. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-159, 21. Fong TL, Shindo M, Feinstone SM, Hoofnagle JH, Di Bisceglie AM. Detection of replicative intermediates of hepatitis C viral RNA in liver and serum of patients with chronic hepatitis C. J Clin Invest 1991;88:1058-1060. 22. Okamoto H, Okada S, Sugiyama Y, Tanaka T, Sugai Y, Akahane Y, Machida A, Mishiro S, Yoshizawa H, Miyakawa Y, Mayumi M. Detection of hepatitis C virus RNA by a two-stage polymerase chain reaction with two pairs of primers deduced from the 5’-noncoding region. Jpn J Exp 1990;60:215-222.
Address requests for reprints to: Ding-Shinn Chen, M.D., Department of Internal Medicine, National Taiwan University Hospital 1, Chang-Te Street, Taipei, Taiwan 10016, Republic of China. Supported by grants from Department of Health, National Science Council: Executive Yuan; and Institute of Biomedical Sciences, Academia Sinica, Republic of China.
1992;103:1322-1327
Hepatitis C and B Viruses in Hepatitis B Surface Antigen-Negative Hepaiocellular Carcinoma JIN-CHUAN WEN-CHEN MING-YANG HSUAN-SHU
SHEU, GUAN-TARN HUANG, LING-NA SHIH, LEE, HUEY-CHI CHOU, JIN-TOWN WANG, PO-HUANG LAI, CHANG-YI WANG, PEI-MING YANG, LEE, and DING-SHINN
CHEN
Department of Internal Medicine, College of Medicine, National Taiwan University Republic of China; and United Biomedical Inc., Lake Success, New York
The relative role of hepatitis C virus and hepatitis B virus in hepatitis B surface antigen-negative hepatocellular carcinoma was evaluated by polymerase chain reaction in 31 patients from Taiwan. Twenty-one were positive for antibody to hepatitis C virus (group 1)and 10 were negative (group 2). Of the group 1 patients, hepatitis C viral RNA was detected in the serum by polymerase chain reaction in 16 and in the liver tissue in 17, whereas hepatitis B viral DNA was found in the liver tissue in only 4, and none were found in the serum. In group 2 patients, hepatitis C viral RNA was detected in the serum of 1 and in the liver tissue of another. In contrast, hepatitis B viral DNA was found in the serum of 4 patients and in the liver tissues of 5. It was concluded that hepatitis C virus plays an important role in hepatocarcinogenesis in hepatitis B surface antigen-negative patients in Taiwan, especially in those who had antibody to hepatitis C virus; in those without antibody to hepatitis C virus, hepatitis B virus might still be associated with the development of hepatocellular carcinoma in a significant proportion of such patients. epatocellular carcinoma (HCC) is one of the most common malignancies in the world, especially in sub-Saharan Africa and Southeast Asia.ln’ Chronic hepatitis B virus (HBV) infection has been well documented to have a major role in the development of this cancer.‘s3 In patients negative for hepatitis B surface antigen (HBsAg), integration of HBV DNA sequences in the HCC tissue has been found.4-7 However, the positive rate reported has been inconsistent.4-8 Recently, the hepatitis C virus (HCV) has been implicated as another major causative agent of HCC in areas where HBV infection is low in prevalence.g-ll In HBV hyperendemic areas such as Taiwan, the prevalence of antibody to HCV (anti-HCV)
H
LEE,
Hospital, Taipei, Taiwan,
has been found to be much higher amongHBsAg-negative HCC patients than among HBsAg-positive HCC patients.” However, the significance of the association of HCV with HCC is difficult to interpret from serologic markers alone because although they are not antigenemic for HBsAg, nearly all of the HBsAg negative HCC patients in Taiwan have been infected by HBV as shown by the presence of antibodies to surface (anti-HBs) or core (anti-HBc) antigen.3 The polymerase chain reaction (PCR), which can detect a single copy of gene, provides a sensitive method to detect the HCV and HBV genome.13-l* To clarify the relative role of HCV and HBV in HBsAg-negative HCCs, we used PCR to detect the HCV RNA and HBV DNA sequences in the serum and liver tissues from 31 HBsAg-negative HCC patients in Taiwan. Patients and Methods Patients The 31 subjects selected for this study were taken from a total of 357 HCC patients seen consecutively in a period of one year and 6 months (July 1988-1990)in our clinic. Of them, 258 were positive for HBsAg and 99 were negative. Among the 99 HBsAg-negative patients, 31 who had frozen liver tissues and serum samples available for analysis were included in this study. The 31 patients ranged from 27 to 78 years old (mean 60), with 19 males and 12 females. Antibodies to HCV were identified in 21 of these 31 cases (67.7%; Table l),15 by standard assays for anti-C160 anti-HCV enzyme-linked immunosorbent assay (Ortho Diagnostic Systems, Raritan, NJ). An additional 6 anti-HCV positive cases were identified by a more sensitive second-generation anti-HCV immunoassay, (UBI HCV EIA; United 0
1992 by the American Gastroenterological 0016-5065/92/$3.00
Association
HCV AND HBV IN HBsAc-NEGATIVE HEPATOMA
October 1992
Table 1. Clinical Data and Results of HCV RNA and HBV DNA in HBsAg-Negative
HCC Patients
1323
Who Were Positive for
Anti-HCV (Group 1) HBV DNA by PCR
HCV RNA by PCR
HBV DNA by Tumor
Se1lllIl
Nontumor
Tllm0r
Nontumor
Southern blot
Tumor Case
Liver
Anti-
Anti-
Size
Primer”
state
HBs
HBc
km)
I
Primer”
I
II
Negative
II
1
CIR
P
N
6.2
P
P
N
N
2
CIR
P
P
2.4
P
P
P
P
Primer”
strandb
I
strandb
P
P
P
P
N
N
Pre-
PE-
Negative
II
Serum
S
S
Core
x
s
s
NonCore
X
Tumor
tumor
N
N
N
N
NN
N
N
N
N
N
N
N
N
N
NN
N
N
N
N
N
3
CIR
P
P
2.0
P
P
P
P
P
P
P
P
N
N
N
N
NN
N
N
N
N
N
4
CIR
P
P
2.9
P
P
P
P
N
P
P
N
N
N
N
N
NN
N
N
N
N
N
P
P
P
N
N
N
N
NN
N
N
N
N
N
P
P
P
P
N
N
N
N
NN
N
P
N
N
N N
5
CIR
P
P
2.4
P
P
N
N
6
CIR
P
P
2.6
P
P
P
P
7
CIR
P
P
2.4
P
P
P
P
P
P
P
P
N
N
N
N
NN
N
N
N
N
8
CIR
N
P
5.5
P
P
P
P
P
P
P
P
N
N
N
N
NN
N
N
N
N
N
9
CIR
P
P
5.4
P
P
P
P
P
P
P
P
N
P
P
N
NN
P
N
N
P
N
10
CIR
P
P
2.7
P
P
P
P
N
P
P
P
N
N
N
P
NN
N
N
N
N
N
11
CIR
P
P
3.6
P
P
P
N
P
P
N
P
N
N
N
N
NN
N
N
N
N
N
12
CIR
N
N
9.1
P
P
N
N
P
P
P
N
N
N
N
NN
N
N
N
N
N
13
CIR
P
P
3.0
P
P
P
P
P
P
P
N
N
N
N
NN
N
N
N
N
N
14
CIR
N
P
2.3
N
N
N
N
N
N
N
N
N
N
NN
N
N
N
N
N
P
N
N
N
N
NP
N
P
N
N
N
N
N
N
N
N
NN
N
N
N
N
N
N
N
N
N
NN
N
N
N
N
N
N
N
N
N
N
N
NN
N
N
N
N
N
P
P
P
N
N
N
N
NN
N
N
N
N
N
N
N
N
N
NN
N
N
N
N
N
N
N
N
N
NN
N
N
N
N
N
15
CIR
N
P
1.5
N
N
N
N
N
N
16
CIR
P
P
2.2
N
N
N
N
P
N
17
CIR
N
N
N
N
N
N
N
N
18
CIR
P
P
4.1
N
N
P
N
P
P
19
CIR
N
P
3.3
P
P
P
P
P
>lO
20
CPH
P
P
3.4
P
P
N
N
N
N
21
CIR
N
P
1.4
P
P
N
N
P
N
P
CIR, cirrhosis; CPH. chronic persistent hepatitis; N, negative: P, positive. “Result of HCV positive strand RNA. Primer 1: primer derived
from 5’noncoding
region. Primer II: primer from nonstructure
region
bResult of negative strand HCV RNA. Only cases with positive strand HCV RNA were tested.
Biomedical, Inc., Lake Success, NY), which incorporated both nonstructural and capsid antigen for antibody capture.lg The 21 HCC patients with seroreactivity to HCV were designated group 1 (Table 1). The remaining 10 HCC patients were negative for antiHCV and were placed into group 2 (Table 2). Sera of the 31 HBsAg-negative HCC subjects were also tested for the presence of anti-HBs and anti-HBc (Ausria-II, Abbott Laboratories, North Chicago, IL). All but 2 patients were positive for either HBV marker, most of them being positive for both (Tables 1 and 2). The 2 patients negative for both antibody
markers of HBV infection were each positive for anti-HCV. Of the 21 anti-HCV positive patients, 20 had liver cirrhosis, whereas only 3 of the 10 negative HCC subjects were cirrhotic (Tables 1 and 2). HCV RNA Serum sample from each patient as well as liver tissues from both tumor and nontumor portion were evaluated by PCR to detect the HCV RNA and HBV DNA sequences. RNA for HCV PCR analysis was extracted from serum as described previously.‘7~‘8 RNA of tissue was isolated from minced
Table 2. Clinical Data and Results of HCV RNA and HBV DNA in HBsAg-Negative Anti-HCV (Group 2)
HCC Patients
HCV RNA by PCR
Who Were Negative for
HBV DNA by PCR HBV DNA by
Serum
TUIIIOI
Nantumor
Tllm0r
Nontumor
Southern blot
Tumor Case
Liver
Anti-
Anti-
Size
state
HBs
HBc
km1
Primer’ I
PrimeP
II
I
Negative II
Primer”
strandb
I
II
Negative strandb
PESerum
s
s
22
CIR
P
P
2.4
N
N
N
N
N
N
NNNNNNNNNN
23
CPH
P
P
11.5
N
N
N
N
N
N
PPP
24
NORM
P
P
>lO
N
N
N
N
N
N
PPP
25
CIR
P
N
>lO
N
N
N
N
N
N
N
26
CPH
P
P
7.0
N
N
N
N
27
NORM
P
P
19.0
N
N
P
N
26
FM
P
P
N
9.4
N
N
P
N
N
P
N
P
P
X
Tumor
tumor
P
N
N
N
NNN
PP
P
N
NN
N
N
NNNN
NN
N
N
NNNN
NN
N
N
NNNN
NN
N
N
N
N
NNN
P
P
6.3
N
N
N
N
N
N
NPNPPPNPPP
P
P
4.5
P
P
N
N
N
N
PNNPNNNNNN
31
CPH
P
P
14.0
N
N
N
N
N
N
P
from 5’noncoding
NonCore
NNN
NORM
NN
region. Primer II: primer from nonstructure
N N N
NNNN
normal; N. negative; P. positive.
bResults of negative strand HCV RNA, only cares with positive strand HCV RNA were tested.
s
NNN
CIR
“Results of positive strand HCV RNA. Primer I: primer derived
s
PP
PPPP P
29
NORM,
x
PPPP
30
CIR. cirrhosis: CPH, chronic persistent hepatitis: FM, fatty metamorphosis;
PWCore
region.
NN
N
N
1324
SHEU ET AL.
liver by a single step extraction thiocyanate-phenol-chloroform.”
GASTROENTEROLOGY
with guanidinium
Positive Strand HCV RNA For the detection of positive strand HCV RNA, RNA from 20 p,L serum or 1.0 pg tissue was reverse transcribed to complementary DNA (cDNA) using 2 sets of antisense primer (primer HCJ296R: SCACTCGCAAGCACCCTATCAGGCAGTACCAJ and primer JK3966R: 5’ATCCCGCTGATGAAGTTCCACATGTGCTTC3’) at 37°C for 90 minutes. Two pairs of primers were used for HCV PCR analysis, one pair from the 5’noncoding regions (primer HCJ004:5’CACTCGCAAGCACCCTATCAGGCAGTACCA3’ and primer HCJ296R as above); the other set from a deduced nonstructural protein encoding region (primer JK3779:5’GAGTGCGCCTCACACCTTCCTTACATCGAAY and primer JK3966R as above). Detection of the HCV amplification products was done on Southern transfers, with 2 labeled 30 oligonucleotides specific for the 5’ noncoding and nonstructural protein sequences (probe HCJ131: YCTGCGGAACCGGTGAGTACACCGGAATTGCY and probe C3821P30:5’GCTCGCCGAGCAGTTCAAGCAGAAGGCCCT3’ respectively). Southern transfer, hybridization, and autoradiography were performed as described previously.17,‘8 Negative Strand HCV RNA To evaluate the status of HCV replication in the liver, liver samples that contained positive strand HCV RNA were further assayed for the presence of negative strand HCV RNA.Z’ RNA from liver tissue was first reverse transcribed with the sense primer derived from the 5’noncoding region (primer HCJ 004 as above). To completely inactivate the reverse transcriptase, the mixture was heated at 98°C for 2 hours. The PCR was then performed as above using the primer pairs derived from 5’noncoding region as above (HCJ 004 and HCJ 296R). HBV DNA PCR assay. For HBV DNA analysis, extraction of serum DNA was done,17,18 and the tissue DNA was extracted with classical method as described before.7 For serum HBV PCR, a pair of nucleotide specific for the core region was used as primer and a 30-mer 32P-labeled oligonucleotide as probe.‘7,‘8 For tissue DNA, 4 pairs of primers specific for core, pre-S, S, and X genes of HBV were used (Table 3). The PCR products were identified after Southern transfer with each of the 4 specific 30-mer oligonucleotides (Table 3). Southern blot analysis. To analyze the status of HBV DNA in the liver, all the tissue DNA samples
Table
3.
Vol. 103, No. 4
Primers and Probes User for Detection DNA by PCR in Tissues
Pre-S region primer 5’GGG TCA 5’ TTC CTG probe 5’GCA AAT S region primer 5’TCT TCT 5’ CTG AAC probe 5’CGG AAA Core region primer 5’ TTT GGT 5’AGA TTC
of HBV
CCA TAT TCT TGG GA 3’ ACT GGC GAT TGG T 3’ CCA GAT TGG GAC TTC AAT CCC AAC 3’
TGT TGG TTC TTC TGG ACT ATC AAG 3’ AAA TGG CAC TAG TAA ACT GAG CCA 3’ TTG CAC CTG TAT TCC CAT CCC ATC 3’
GTC TTT CGG AGT GTG GAT TCG CAC 3’ CCG AGA TTG AGA TCT TCT GCG ACG 3’
probe 5’GAC CAC CAA ATG CCC CTA TCC TAT CAA CAC 3’ X region primer 5’ATG GCT GCT AGG CTG TGC TGC CAA CTG GAT 3’ 5’ CAA GAG TCC TCT TAT GTA AGA CCT TGG GCA 3’ probe 5’ TGC ACT TCG CTT CAC CTC TGC ACG TCG CAT 3’
from the 31 patients were also subjected to Southern blot analysis. Ten micrograms of purified DNA was digested with Hind III, EcoRI, BamHI or StyI, and electrophoresed on 0.8% agarose gel. Southern transfer and hybridization using a 32P-labeled whole genomic HBV DNA as probe was performed as described previously.7 Results HCV RNA Positive stranded HCV RNA. Among the 21 anti-HCV positive group 1 patients, positive strand HCV RNA was detected in the serum in 16 (76.2%) by both sets of HCV primers. Positive stranded HCV sequences were also identified in the liver tissues of 17 (80.9%) of the group 1 patients, 12 in the HCC part and 16 in the nontumor part (Table 1,Figure 1).HCV RNA could be found in the serum, tumor, and nontumor liver tissues of 10 anti-HCV positive cases, in the serum and tumor alone of only 1 case, and in the sera and nontumor liver tissues alone of 3 patients, including those of case 12 who were negative for all HBV serological markers. There were 2 anti-HCV positive patients with no HCV sequences detectable by PCR. HCV RNA was found in only 1 of the 10 anti-HCV negative serum samples of group II (Table 2, case 30), and none could be found in the tissue samples of that individual. Only 1 group 2 patient was positive for HCV RNA in the liver tissues, with HCV sequences
October 1992
012345678
HCV AND HBV IN HBsAG-NEGATIVE
0:
HEPATOMA
1325
012345678
Figure 1. Detection of HCV RNA in the liver tissues by PCR using 5’noncoding region as primer. Arrow indicates 293 base pairs. Lane 0: &xDNA/HaeJII fragment; lanes Z-3: 5 X lo’, 5 X 10,and 5 copies of cloned HCV cDNA fragment; lane4: negative control; lanes 5 and 6: patient 8 in group 1,nontumor and tumor part; lanes7 and 8:patient 14 in group 1,nontumor and tumor part. (A)Gel electrophoresis after PCR amplification showing positive bands in lanes 1, 2, 5, and 6. Lane 6 is faintly visible. (B)Autoradiography showing positive bands in lanes 1, 2, 3, 5, and 6.
identified in the tumor and nontumor liver tissue (case 27). Altogether, among the 31 HBsAg-negative HCC patients of group 1 and 2, HCV positive strand RNA was detected in either the serum or liver tissue in 20 (64.5%) and in the tumor and/or nontumor tissues of 18 (58.1%). The primer sets in PCR for detection of HCV RNA displayed a slight difference in sensitivities. Both sets of the primers amplified HCV sequences in all 17 of the PCR positive serum samples; while the 5’noncoding region primers detected HCV RNA in all 17 of the PCR-positive nontumor tissue samples, the nonstructural protein region primers distinguished 12 of the 17; and the 5’noncoding region primers identified 13 tumor samples as PCR-positive, whereas the nonstructural protein set amplified only 10 of these tumor samples (Tables 1 and 2). Negative strand HCV RNA. In group 1 patients who had positive strand HCV RNA in the liver tissue, the negative strand RNA was detected in 10 of the 12 tumor specimens (83.3%) and in 13 of the 16 nontumor counterparts (81.3%) (Table 1). In group 2 patients, the negative strand HCV RNA was also found in the liver tissues (both the tumor and nontumor part) of the only patient whose tissue had positive strand HCV RNA (case 27, Table 2). HBV DNA PCR assay. By the HBV PCR assay, none of the 21 anti-HCV positive group 1 patients had HBV DNA in the serum (Table 1). However, 4 (19.0%)
were found to have HBV DNA sequences in the liver tissue, 3 in the nontumor portion and 2 in the tumor portion with one (case 9) in both HCC and nontumor part. Of the 4 patients positive for HBV DNA in the tissue, 3 also had HCV RNA in the serum and in the liver tissues, whereas the remaining one had no HCV sequence in either the serum or liver tissue. Of the group 2 anti-HCV negative patients, 4 (40%) were found to have HBV DNA in the serum. HBV DNA sequences were also identified in the liver tissues in 5 cases (50%) with 3 in both tumor and nontumor part and 2 in the tumor part alone (Figure 2). Among the 4 patients who had HBV DNA in the serum, 2 (cases 23 and 24) had HBV DNA in both the tumor and nontumor part detected by all 4 HBV primer sets (Table 2); another one (case 30) had HBV DNA in the tumor part detectable only by primers for HBV core sequences. The remaining one (case 31) had no HBV sequences in the liver tissue. On the other hand, 2 patients (cases 25 and 29) who had no HBV DNA in their serum had detectable HBV sequences in the liver tissues (1 in the tumor alone and the other in both the tumor and nontumor portions). Thus persistent HBV infection as evidenced by HBV DNA in either serum or liver tissue was present in 6 (60%) of these HBsAg-negative anti-HCV negative patients. Overall, among the 31 HBsAg-negative HCC patients of groups 1 and 2, HBV DNA sequences were detected in the liver tissue of 9 cases (29.0%) and in the serum and/or tissues of 10 (32.3%). Southern blot analysis. By Southern blot analysis, only one group 1 patient (case 9; Table 1) and 3
1326
SHEU ET AL.
0123456
GASTROENTEROLOGY
Vol. 103. No. 4
0123456
Figure 2. Detection of HBV DNA in the liver tissue by PCR using HBV core sequence as primer. Arrow indicates 190 base pairs. Lane 0: 4xDNA/HaeIII fragment; lanes 2 and 2: patient 23 in group 2, tumor and nontumor part; lanes 3 and 4: patient 30 in group 2, tumor and nontumor part; lanes 5 and 6: patient 31 in group 2, tumor and nontumor part. (A)Gel electrophoresis after PCR amplification showing positive bands in lanes 1 and 2. (B) Autoradiography showing positive bands in lanes 1-3.
group 2 patients (Cases 23,24,and 29; Table 2) had detectable HBV genome in the tumor part and no HBV DNA was found in the nontumor part. All the HBV DNA was present in integrated form. Three of the patients showed only one discrete band, whereas the remaining one (case 23;Table 2)showed multiple discrete bands. Discussion We found that HCV RNA was detected in either the serum, tumor and/or nontumor tissues of 20 of the 31 non-HBsAg antigenemic HCC patients, associating persistent HCV infection with 64.5% of those cases. The correlation of chronic HCV infection with HCC was more marked in the 21 HBsAg-negative anti-HCV positive patients. HCV RNA was found in either the serum, liver, or tumor samples of 18 of these 21 (85.7%). Although the PCR method could not differentiate between tissue derived and serum derived HCV, we found that the majority (>80%) of group 1 patients who had positive strand HCV RNA in the liver tissue also had negative strand HCV RNA in the liver tissue. Indeed the presence of the replicative form of HCV RNA proved that active HCV infection is present in these patients. Moreover, the low prevalence of HBV sequence in these 21 HCC patients is in favor of chronic HCV infection to be etiologically important for HCC in the HBsAg negative anti-HCV positive patients. On the other hand, 4 patients in this group also had HBV DNA sequences in the tissue; 3 of them also had concurrent HCV RNA in the tissue and serum. This finding indicates that simultaneous persistent infection of HCV and HBV
might contribute to the hepatocarcinogenesis in some of these HBsAg negative HCC patients. In the group 2 anti-HCV negative patients, HCV RNA was uncommonly found, being present in the serum of only one and in the liver of another. In contrast, HBV DNA was more frequently found in either the serum or tissue (60%).By Southern blot hybridization, 3 patients in this group had an integrated form of HBV sequences in the tumor part. This finding indicated that in these HBsAg negative antiHCV negative HCC patients, HBV might still play a more important role than HCV in the hepatocarcinogenesis. HBV DNA was not detectable in the patient (Case 27;Table 2) who had both positive and negative strands of HCV RNA in the liver tissue, suggesting HCV might still be associated with the development of HCC even in the absence of circulating anti-HCV antibody. By Southern blot hybridization, most investigators reported that only a minority of the HBsAg negative HCC had HBV DNA integration in the tumor cells, although a very high positive rate has been describede4s5 Consistent with our previous data,7 we again showed a rather low positive rate in this setting of HCC patients. Although the positive rate of HBV DNA detected by Southern blot analysis is much lower than those by PCR as shown in our study in the tumor tissue, the fact that they all appear in integrated form and not in the nontumor part suggests that the HBV is present in more copies in the tumor part and might well represent the clonal expansion of HBV-integrated hepatocytes in the cancer tissue. In both groups of HCC patients (Tables 1 and 2), the primer pair corresponding to the 5’ noncoding se-
October 1992
quence of the HCV genome was more sensitive than the nonstructural protein region primers for the detection of HCV RNA. This observation is in agreement with nucleic acid sequence data showing the 5’ noncoding region to be more conserved than the nonstructural region.” We conclude that HCV plays an important role in the hepatocarcinogenesis in HBsAg-negative subjects in Taiwan, especially in those positive for antiHCV; in the HBsAg negative anti-HCV negative HCC patients, chronic HBV infection might still be closely associated with the development of HCC. References 1 Linsell DA, Higginson J. The geographic
2.
3
4.
5.
6.
7.
8.
9.
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Address requests for reprints to: Ding-Shinn Chen, M.D., Department of Internal Medicine, National Taiwan University Hospital 1, Chang-Te Street, Taipei, Taiwan 10016, Republic of China. Supported by grants from Department of Health, National Science Council: Executive Yuan; and Institute of Biomedical Sciences, Academia Sinica, Republic of China.